Primer Extension
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RNA isolation
Primer Labeling
- To 0.5pmole primer (0.5µl 10pmoles/µl) add 1µl 10x T4 polynucleotide kinase buffer from Roche kit.
- Add 2.5µl 32 P-γ-labelled dATP (5µl if using 32 P-γ-labelled dATP past it''s first half life).
- Add DEPC treated water to make up to 9µl (4.5µl if using 10pmoles/µl primer).
- Add 1µl T4 polynucleotide kinase from same kit.
- Incubate for 30 minutes at 37°C.
- Remove top and bottom cap from NAP-5 column (Amersham-Pharmacia Biotech) to drain of storage liquid.
- Fill up with DEPC treated water 3 times and allow to drain.
- Add 500µl DEPC treated water to primer labeling reaction.
- Add primer labeling reaction to NAP-5 column and allow cold flow through to drain off.
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Add 1ml of DEPC treated water and collect eluate with purified hot primer.
Reverse transcription with Superscript II from radioactive primer
- Take 30µg of RNA for highly expressed genes or 50µg for poorly expressed genes and adjust volume to 40 µl.
- Add 60µl of the eluate from the purification of labeled primer.
- Add 10µl (1/10 volume) of 3M sodium acetate.
- Add 250µl of 95% ethanol.
- Incubate overnight at -70°C.
- Spin for 30 minutes at full speed.
- Remove and discard supernatant carefully.
- Add 500µl 75% ethanol.
- Spin 10 minutes at full speed.
- Remove and discard supernatant carefully.
- Resuspend in 30µl of DEPC treated water.
- Incubate at 90�C for 3 minutes.
- Cool slowly to under 30°C.
- Add 18.5µl of DEPC treated water.
- Add 20µl of undefined Superscript II buffer.
- Add 10µl 0.1 M DTT.
- Add 20µl dNTP-mix for primer extension.
- Add 1µl of RNasin (RNase-inhibitor, Promega).
- Add 0.5µl Superscript II reverse transcriptase.
- Incubate at 42�C for 90 minutes.
- Add 1µl 0.5M EDTA and mix.
- Add 0.5µl RNaseA (Promega) and mix.
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Incubate at 42°C for 10 minutes.
Purification by Phenol/Chloroform extraction
- Add 100µl Phenol to the reaction, mix by vigerous shaking.
- Spin 10 minutes at full speed to separate phases.
- Transfer the aquaous (top) phase to a new tube with 125µl 50:50 Phenol:Choloroform.
- Add 25µl dH2 O to the tube containing the Phenol phase.
- Transfer the aquaous phase to the tube with Phenol:Chloroform, mix by vigerous shaking.
- Spin 10 minutes at full speed to separate phases.
- Transfer the aquaous (top) phase to a new tube with 150µl Choloroform.
- Add 25µl dH2 O to the tube containing the Phenol:Cholorofom phase.
- Transfer the aquaous phase to the tube with Chloroform, mix by vigerous shaking.
- Spin 10 minutes at full speed to separate phases.
- Transfer the aquaous (top) phase to a new tube.
- Add 25µl dH2 O to the tube containing the Chloroform phase.
- Transfer the aquaous phase to the tube with the aquaous phase from previously.
- Add 17.5µl (1/10th volume) 3M Sodium-Acetate and mix.
- Add 550µl (3 volumes) 95% ethanol.
- Leave at -70°C for 15 minutes or overnight.
- Spin 30minutes at full speed.
- Remove supernatant, and add 500µl 75% ethanol.
- Spin for 10minutes.
- Remove supernatant and airdry until no ethanol is left in tube.
- Dissolve in 6µl dH2 O.
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Add 4µl stop solution from Amersham manual sequncing kit.
Load and run on a manual sequencing gel along with a manual sequencing reaction preferably done on the relevant gene with the same primer according to manufactureres directions. Dry the gel and expose X-ray film to it for 1 to 2 days.
Solutions
Production and purification of labeled cDNA
- dNTP-mix for primer extension
- 10µl 100mM dATP, 10µl 100mM dCTP, 10µl 100mM dGTP, 10µl 100mM dTTP, 10µl DEPC-treated water .
